Dna replication factors

ABSTRACT

A DNA polymerase reaction system which provides high DNA polymerase activity even at a high temperature and at a high salt concentration. A DNA polymerase reaction system that is constructed from a DNA polymerase, a clamp, and a clamp loader without intein sequence, the DNA polymerase being from  Pyrococcus horikoshii , a hyperthermophilic archaeon.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to two DNA replication factors capable ofenhancing DNA polymerase activity from Pyrococcus, and to a DNApolymerase reaction system utilizing such factors.

2. Background Art

DNA polymerase is an enzyme useful for DNA sequencing reaction,polymerase chain reaction (PCR), radioactive labeling of DNA, in vitrosynthesis of a mutated gene, and the like. DNA polymerases currentlyknown can be generally classified into six families based on their aminoacid sequence homology. Among them, DNA polymerases usually used asreagents in gene manipulation experiments belong to Family A polymerasessuch as typical E. coli DNA pol I and thermophilic bacterium Thermusaquaticus DNA polymerase (i.e., Taq DNA polymerase), and Family Bpolymerases such as typical T4 phage DNA polymerase. Various DNApolymerases having different optimum temperatures have been discoveredfrom bacteria as well as from animals and plants. However, many of them,derived from mesophilic organisms and thus having low thermostability,are not suitable, for example, for PCR comprising heat denaturation oftemplate DNA at 94° C. or over.

Enzymes from thermophilic bacteria, such as Taq DNA polymerase, arecommercially available as thermostable DNA polymerases. However, all ofthem lack 3′-5′ proofreading exonuclease activity, resulting in highererror rates during polymerase reactions such as PCR, and hence are notsuitable for PCR with high fidelity and the like. Further, type Benzymes that are thermostable and have 3′-5′ proofreading exonucleaseactivity, are isolated from hyperthermophilic archaea such as Pyrococcusand Thermococcus, and are commercially available. However, they have lowprimer extension activity and so are not suitable for PCR forlong-strand DNA.

Examples of PCR techniques developed so far include conventional PCRusing commercially available thermostable Pol A or Pol B, and isothermalPCR using φ29 DNA polymerase having high strand-displacement activity.The region replicable by the conventional Pol A or Pol B enzymes isshort, the maximum being about 10 kb. In addition, the synthesis rate isas low as 30 b/sec. On the other hand, when φ29 DNA polymerase is used,PCR can be conducted at ambient temperature, so no expensive apparatusfor amplification reaction is required and the procedure is simple.However, the use of random primers causes replicated regions to berelatively short, so that it is difficult to synthesize or produce along DNA strand. Further, it is difficult to amplify DNA directly fromthe blood, body fluid, etc., by using the above conventional enzymes.This is because any of those conventional enzymes exhibits a reducedactivity of DNA synthesis at a high salt concentration, and hencedesalting is required to lower the salt concentration in the reactionsolution.

The present inventors, for the first time, discovered a DNA polymerasefrom Pyrococcus horikoshii, which is thermostable and has 3′-5′proofreading exonuclease activity, and the gene thereof (JP Patent No.3015878). Moreover, we successfully improved the DNA polymerase activitydramatically by removing an intein sequence from a large subunit of theDNA polymerase (JP Patent Publication (Kokai) No. 2001-299348A).Further, this DNA polymerase has a unique property that its primerextension activity becomes higher as the primer length is longer.However, this DNA polymerase also exhibits a reduced activity at a highsalt concentration, although its activity is high at a low saltconcentration.

Under these circumstances, the object of the present invention is toconstruct a new DNA polymerase reaction system utilizing a DNApolymerase, which is highly thermostable, has 3′-5′ exonuclease activityby which mistakes occurring in a newly extended DNA strand arecorrected, and also exhibits a high primer extension activity, whereinthe high DNA polymerase activity is exerted even at a high saltconcentration. Thereby, a novel technology is provided wherein along-strand DNA region having a length of several Mb can be quicklyreplicated even under a high salt concentration without pretreatment.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned problems, the present inventorsfocused on the hyperthermophilic archaeon Pyrococcus horikoshii, whichgrows at a temperature from 90 to 100° C., and discovered, from its genesequences, genes expected to encode a clamp and a clamp loader, whichare replication factors interacting with DNA polymerase to enhance DNApolymerase activity. Further, the clamp and the clamp loader wereproduced using E. coli from the discovered genes, and then it wasconfirmed that these proteins are stable at an elevated temperature (85°C. or higher), and enhance a primer extension activity of the DNApolymerase even at a concentration of sodium chloride as high as 0.2 M,thus the present invention having been completed.

In summary, the present invention comprises:

-   (1) A method for synthesizing a DNA, in which a primer DNA is    extended by a DNA polymerase to synthesize the DNA complementary to    a template DNA using deoxynucleotide triphosphates as substrates,    wherein the enzyme reaction system comprises the DNA polymerase from    Pyrococcus horikoshii and the two protein complexes of the    following a) and b):-   a) a protein complex composed of three molecules of a subunit and    having a clamp function, the subunit being a protein comprising the    amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having    at least 90% identity with the amino acid sequence of SEQ ID NO: 8;-   b) a protein complex composed of one molecule of a large subunit and    four molecules of a small subunit and having a clamp loader    function, wherein the large subunit is a protein comprising the    amino acid sequence of SEQ ID NO: 10 or an amino acid sequence    having at least 90% identity with the amino acid sequence of SEQ ID    NO: 10, and wherein the small subunit is a protein comprising the    amino acid sequence of SEQ ID NO: 14 or an amino acid sequence    having at least 90% identity with the amino acid sequence of SEQ ID    NO: 14.-   (2) The method of the above (1), wherein said enzyme reaction system    does not contain ATP.-   (3) The method of the above (1) or (2), wherein said enzyme reaction    system contains sodium chloride at a concentration of from 0 to 200    mM/L or >0 to 200 mM/L.-   (4) A reagent kit for synthesizing a DNA, wherein the kit comprises    a DNA polymerase from Pyrococcus horikoshii and the two protein    complexes of the following a) and b):-   a) a protein complex composed of three molecules of a subunit and    having a clamp function, the subunit being a protein comprising the    amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having    at least 90% identity with the amino acid sequence of SEQ ID NO: 8;-   b) a protein complex composed of one molecule of a large subunit and    four molecules of a small subunit and having a clamp loader    function, wherein the large subunit is a protein comprising the    amino acid sequence of SEQ ID NO: 10 or an amino acid sequence    having at least 90% identity with the amino acid sequence of SEQ ID    NO: 10, and wherein the small subunit is a protein comprising the    amino acid sequence of SEQ ID NO: 14 or an amino acid sequence    having at least 90% identity with the amino acid sequence of SEQ ID    NO: 14.-   (5) The reagent kit of the above (4), wherein the kit is used for    PCR and optionally contains written instructions, primers and/or    ancillary reagents used for PCR.-   (6) A protein comprising the amino acid sequence of SEQ ID NO: 8, or    an amino acid sequence having at least 90% identity with the amino    acid sequence of SEQ ID NO: 8, wherein, when three molecules of the    protein subunit form a protein complex, the complex has a clamp    function.-   (7) A protein comprising the amino acid sequence of SEQ ID NO: 10 or    an amino acid sequence having at least 90% identity with the amino    acid sequence of SEQ ID NO: 10, wherein, when one molecule of the    protein subunit forms a protein complex with four molecules of a    small subunit comprising the amino acid sequence of SEQ ID NO: 14 or    an amino acid sequence having at least 90% identity with the amino    acid sequence of SEQ ID NO: 14, the complex has a clamp loader    function.-   (8) A protein comprising the amino acid sequence of SEQ ID NO: 14 or    an amino acid sequence having at least 90% identity with the amino    acid sequence of SEQ ID NO: 14, wherein, when four molecules of the    subunit form a protein complex with one molecules of a large subunit    comprising the amino acid sequence of SEQ ID NO: 10 or an amino acid    sequence having at least 90% identity with the amino acid sequence    of SEQ ID NO: 10, the complex has a function as a clamp loader.-   (9) A DNA encoding of the protein of any one of the above (6) to    (8).-   (10) A DNA of the following (i) or (ii):-   (i) a DNA comprising the nucleotide sequence of SEQ ID NO: 7, SEQ ID    NO: 9, or SEQ ID NO: 13;-   (ii) a DNA hybridizing with a DNA comprising a nucleotide sequence    complementary to the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO:    9, or SEQ ID NO: 13 under stringent conditions.-   (11) A recombinant vector comprising the DNA of the above (9) or    (10).-   (12) A host cell comprising the recombinant vector of the above (11)    is introduced therein.

According to the present invention, two novel DNA replication factorsderived from Pyrococcus horikoshii, that is, a clamp and a clamp loader,is provided. Thus, an enzyme reaction system with which a long DNAstrand is efficiently and accurately replicated by a DNA polymeraseunder conditions of a high temperature and a high salt concentration canbe constructed. This enzyme reaction system is particularly useful forLong-PCR and the like, and new approaches for gene sequence analysisusing such an enzyme reaction system can be developed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the gene sequence of the large subunit of the DNApolymerase D (SEQ ID NO: 1). Underlined is the intein sequence.

FIG. 2 illustrates the amino acid sequence of the large subunit of theDNA polymerase D (SEQ ID NO: 2). Underlined is the intein sequence.

FIG. 3 illustrates the gene sequence of the small subunit of the clamploader (RFC) (SEQ ID NO: 11). Underlined is an intein sequence.

FIG. 4 illustrates the amino acid sequence of the small subunit of theclamp loader (RFC) (SEQ ID NO: 12). Underlined is an intein sequence.

FIG. 5 illustrates the results determined for optimum pH of the DNApolymerase of the present invention.

FIG. 6 illustrates the results determined for optimum Mg2+ concentrationof the DNA polymerase of the present invention.

FIG. 7 illustrates the residual activity of the DNA polymerase of thepresent invention determined after heat treatment.

FIG. 8 illustrates the results tested for primer extension activity ofthe DNA polymerase of the present invention. For Lanes 1 to 3, Lanes 4to 6, and Lanes 7 to 9, 15 mer primers, 34 mer primers, and a 50 merprimers were used, respectively. The reaction was constructed, for Lanes1, 4, and 7, for 2 min in the presence of the enzyme; and for Lanes 2,5, and 8, for 10 min in the presence of the enzyme. Lanes 3, 6, and 9represent control experiments, in which no enzyme was added to thereaction system.

FIG. 9 illustrates the results tested for 3′-5′ exonuclease activity ofthe DNA polymerase of the present invention. A 50 mer oligonucleotidewas used as a substrate. For Lane 1, the reaction was conducted for 30min in the presence of the enzyme, and Lane 2 represents a control withno enzyme.

FIG. 10 illustrates the results determined by SDS-PAGE for the molecularweight of PCNA of the present invention. Lane 1 shows molecular markers,and Lane 2 shows purified PCNA.

FIG. 11 illustrates the results determined by SDS-PAGE for the molecularweight of RFC complex of the present invention. Lane 1 shows molecularmarkers, and Lane 2 shows purified RFC complex, wherein L indicates thelarge subunit and S indicates the small subunit.

FIG. 12 illustrates the results of the pull-down assay of PCNA by RFCcomplex of the present invention. The interaction between RFC complexand PCNA was analyzed in the presence or absence of ATP by SDS-PAGE.

FIG. 13 illustrates the results tested for the effect of RFC complex andPCNA on enhancement of the activity of DNA polymerase D. ATP was used atthe concentration of 10 mM.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Each of the DNA polymerase, the clamp (proliferating cell nuclearantigen, PCNA), and the clamp loader (replication factor, RFC) used inthe present invention is a protein complex that is derived fromPyrococcus (JCM Accession Number 9974) and composed of multiplesubunits.

The aforementioned DNA polymerase is a thermostable heterodimer enzymecomposed of a small subunit and a large subunit without intein sequence,and has 3′-5′ exonuclease activity and to DNA polymerase activity. Thisenzyme also has a primer extension activity dependent on primer lengthand has a property that the primer extension activity increases when theprimer length is over 30 mer. A DNA polymerase composed of the smallsubunit and the large subunit without intein sequence is described inthe previous patent application filed by the present inventors (JPPatent Application No. 2000-116257 (JP Patent Publication (Kokai) No.2001-299348A)), and hence the enzyme without intein sequence itself iswell known.

The invention of the previous application is based upon the fact that itwas found for the first time that there is an intein sequence in thelarge subunit of the thermostable heterodimer enzyme, which is derivedfrom Pyrococcus horikoshii and has DNA polymerase activity as well as3′-5′ exonuclease activity and that, when the intein sequence isremoved, the primer extension activity of the enzyme is significantlyimproved. The amino acid sequence of the small subunit of thisthermostable heterodimer enzyme from Pyrococcus horikoshii and thenucleotide sequence of the gene thereof are shown in SEQ ID NO: 6 andSEQ ID NO: 5, respectively. The amino acid sequence of the large subunitand the nucleotide sequence of the gene thereof are shown in SEQ ID NO:2 and SEQ ID NO: 1 respectively, as well as in FIG. 2 and FIG. 1respectively. The amino acid sequence of the intein portion correspondsto the region from amino acids 955 to 1120, and the nucleotide sequencethereof to the region from nucleotides 2863 to 3360 (the intein sequenceis underlined in FIG. 2 and FIG. 1).

The amino acid sequence of the large subunit with no intein portion andthe nucleotide sequence corresponding thereto are shown in SEQ ID NO: 4and SEQ ID NO: 3, respectively. This enzyme is extremely thermostableand, even after treated at 85° C. for 1 hr., it retains 50% activitywhen compared to that before treatment. Additionally, after treated at90° C. for 1 hr., it retains 20% activity. The optimum pH of theactivity is pH 8.5.

The clamp (hereinafter, also referred to as PCNA) is a ring-shapedprotein complex composed of plural subunits that tethers the DNApolymerase to a primed-template DNA during DNA replication. The clamploader is a protein complex composed of plural subunits that opens andcloses this clamp.

The clamp (PCNA) and the clamp loader (RFC), which are replicationfactors interacting with the DNA polymerase, a heterodimer, to enhancethe DNA polymerase activity, can be obtained from the gene sequences ofPyrococcus horikoshii described above by the following procedures.

The gene of the clamp (PCNA) is amplified by PCR and extracted, which isthen inserted into a vector such as a protein expression plasmid. Theresulting plasmid is introduced into a host microorganism such as E.coli, which is then cultured to produce the clamp (PCNA). The producedclamp (PCNA) is heated and subsequently subjected to isolation andpurification by column chromatography. Thus, the clamp (PCNA) isobtained. The purified PCNA has been revealed to be a homotrimercomposed of the subunit having a molecular weight of 28 kDa. The aminoacid sequence of the subunit and the nucleotide sequence correspondingthereto are shown in SEQ ID NO: 8 and SEQ ID NO: 7, respectively.

On the other hand, the clamp loader (RFC) from Pyrococcus horikoshii iscomposed of two subunits having different molecular weights. The subunithaving a larger molecular weight has an intein sequence, and the aminoacid sequence of this subunit and the nucleotide sequence correspondingthereto are shown in SEQ ID NO: 12 and SEQ ID NO: 11 respectively, aswell as in FIG. 4 and FIG. 3 respectively (the intein sequences areunderlined in FIG. 4 and FIG. 3). In the present invention, each of theregions upstream and downstream of the intein sequence in the DNAencoding the subunit (SEQ ID NO: 11; FIG. 3) is amplified by PCR, theresulting two DNA fragments are used as templates for amplification byOverlap PCR. Thus, the DNA encoding the subunit from which the inteinsequence is removed is obtained. Hereinafter, the subunit without inteinsequence is referred to as small subunit, and the subunit thatoriginally contains no intein sequence is referred to as large subunit.

The DNA encoding the large subunit and the DNA encoding the smallsubunit without intein sequence, which subunits are of the clamp loader(RFC), are inserted into a single expression vector or two separateexpression vectors. A host microorganism is then transfected with theresulting vector or vectors for coexpression. Further, the resultingproteins can be heat-treated and subsequently subjected to purificationand isolation by column chromatography. Herein, the coexpression refersto the phenomenon wherein two DNAs are expressed in a hostsimultaneously and respective proteins corresponding to those DNAs areproduced therein. The clamp loader having the activity recited in thepresent invention can be obtained by coexpression. The clamp loader thusobtained is composed of the large subunit having a molecular weight of54 kDa and the small subunits containing no intein sequence and having amolecular weight of 38 kDa. The clamp loader is a heteropentamer, whichis composed of one molecule of the large subunit and four molecules ofthe small subunit without intein sequence. The amino acid sequence ofthe 54 kDa large subunit and the corresponding nucleotide sequence areshown in SEQ ID NO: 10 and SEQ ID NO: 9, respectively. The amino acidsequence of the 38 kDa small subunit without intein sequence and thecorresponding nucleotide sequence are shown in SEQ ID NO: 14 and SEQ IDNO: 13, respectively.

Each of the subunits composing the DNA polymerase, the clamp, or theclamp loader of the present invention includes a polypeptide comprisingan amino acid sequence having at least 80 or 85%, preferably at least90%, more preferably at least 95, 96, 97, or 98%, still more preferablyat least 99% identity with the respective amino acid sequence of SEQ IDNO: 2, 4, or 6 (DNA polymerase subunit), SEQ ID NO: 8 (PCNA subunit), orSEQ ID NO: 10, 12, or 14 (RFC subunit). In particular, each of thesubunits composing the above DNA polymerase, the clamp, and the clamploader of the present invention is not limited to a polypeptide thatcomprises the amino acid sequence of each of SEQ ID NOS described above,but also includes a polypeptide that comprises an amino acid sequence inwhich one or more, preferably one or several, amino acids are deleted,substituted or added in any of these amino acid sequences, as long asthe DNA polymerase activity, or the clamp function or clamp loaderfunction, is still provided when it forms a respective protein complexas described above.

In the present invention, the term “several” refers to an integer of,for example, from 2 to 20, preferably from 2 to 15, more preferably from2 to 10, from 2 to 9, from 2 to 8, from 2 to 7, from 2 to 6, from 2 to5, from 2 to 4, or from 2 to 3.

Herein, in regard to amino acid substitution, amino acid side chains aredifferent one another in terms of chemical properties such ashydrophobicity and electric charge or structural properties. Some highlyconservative relationships, wherein the three-dimensional structure(also referred to as conformation) of the entire polypeptide is notessentially affected, are known from experience or actualphysicochemical measurements. Substitution between amino acids in thepresent invention may be conservative substitution between amino acidswhich are similar to each other in terms of chemical or structuralproperties, or may be non-conservative substitution between amino acidswhich are different from each other in terms of such properties. Aminoacids can be classified based on similarities in chemical and structuralproperties into the following groups.

In the hydrophobic amino acid groups, alanine (Ala), leucine (Leu),isoleucine (Ile), valine (Val), methionine (Met), and proline (Pro) areincluded.

In the polar amino acid group, serine (Ser), threonine (Thr), glycine(Gly), glutamine (Gln), asparagine (Asn), and cysteine (Cys) areincluded.

In the aromatic amino acid group, phenylalanine (Phe), tyrosine (Tyr),and tryptophan (Trp) are included. In the acidic amino acid group,glutamic acid (Glu) and aspartic acid (Asp) are included.

In the basic amino acid group, lysine (Lys), arginine (Arg), andhistidine (His) are included.

Examples of conservative substitution include the substitution betweenthe following amino acids: glycine (Gly) and proline (Pro); glycine(Gly) and alanine (Ala) or valine (Val); leucine (Leu) and isoleucine(Ile); glutamic acid (Glu) and aspartic acid (Asp); glutamine (Gln) andasparagine (Asn); cystein (Cys) and threonine (Thr); threonine (Thr) andserine (Ser) or alanine (Ala); lysine (Lys) and arginine (Arg), etc.

Moreover, the DNA encoding each of the subunits composing the clamp orclamp loader of the present invention includes DNA comprising anucleotide sequence having at least 80 or 85%, preferably at least 90%,more preferably 95, 96, 97, or 98%, still more preferably 99% identitywith the respective nucleotide sequence of SEQ ID NO: 7 (PCNA subunit)or SEQ ID NO: 9, 11, or 13 (RFC subunit). In particular, the DNAencoding each of these subunits in the present invention is not limitedto a DNA that comprises the nucleotide sequence of each of SEQ ID NOSdescribed above, but also includes a DNA that encodes a proteincomprising the amino acid sequence of each of SEQ ID NOS described aboveor a DNA that encodes a protein comprising an amino acid sequence inwhich one or more, preferably one or several, amino acids are deleted,substituted, or added in any of such amino acid sequences, wherein, whenit forms each of the protein complexes described above, the DNA canencode a subunit capable of providing the respective functions.

The identity ranges and values for polynucleotide and polypeptidesequences herein include all intermediate subranges and values, forexample, 80, 81, 82.5, 90, 92.5, 95, 95.1, 95.2, 95.3, 95.5, 99.65 or99.75% identity to the corresponding sequence.

The DNA encoding each of the subunits composing the clamp or clamploader of the present invention not only include DNA comprising therespective nucleotide sequence of SEQ ID NO: 7 (the PCNA subunit) or SEQID NO: 9, 11, or 13 (the RFC subunits) but also includes DNA comprisinga mutated nucleotide sequence of any of these nucleotide sequences basedon degeneracy of the genetic code. For example, the most suitable codonfor a species different from Pyrococcus horikoshii can be selected basedon the degeneracy of the genetic code for incorporation of the DNA intothe cells of the species. Such a mutated nucleotide sequence refers to anucleotide sequence comprising a different codon(s) for a certain aminoacid(s).

Alternatively, the DNA encoding each of the subunits composing the clamp(PCNA) or clamp loader (RFC) of the present invention may include ananalogue or homologue of the DNA comprising the nucleotide sequence ofeach SEQ ID NO described above, and is functionally equivalent thereto.As used herein, the term “functionally equivalent” means that thepolypeptide encoded by the above analogue or homologue of the DNA hasbiological and/or biochemical functions that are equivalent to those ofthe peptide encoded by the DNA consisting of the nucleotide sequence ofthe respective SEQ ID NOS. More specifically, such a polypeptide has thefunction of each subunit composing the clamp (PCNA) or the clamp loader(RFC). Such a DNA includes a DNA that hybridizes with a DNA consistingof a nucleotide sequence complementary to the nucleotide sequence of anyof SEQ ID NOS described above under stringent conditions. The stringentconditions refer to conditions under which specific hybrid is formedwhile non-specific hybrid not formed. The stringency comprises high orlow stringency, and preferred is high stringent condition. In the lowstringent condition, wash after hybridization is carried out, forexample, using a solution of 5×SSC and 0.1% SDS at 42° C., andpreferably using a solution of 5×SSC and 0.1% SDS at 50° C. In the highstringent condition, wash after hybridization is carried out, forexample, using a solution of 0.1×SSC and 0.1% SDS at 65° C. Therefore,even when partial alterations of the full length nucleotide sequence ofany SEQ ID NO described above can be generated by various artificialmanipulations such as site-directed mutagenesis, random mutation usingmutagens, or mutation, deletion or ligation of a DNA fragment cleaved bya restriction enzyme, the DNA variant which hybridizes with a DNAcomprising a nucleotide sequence complementary to the nucleotidesequence of any of SEQ ID NOS described above under stringentconditions, and encodes a polypeptide having the function of each of thesubunits composing the above clamp (PCNA) or the clamp loader (RFC) isincluded in the DNA encoding each subunit composing the clamp (PCNA) orthe clamp loader (RFC) according to with the present invention,regardless of differences from the nucleotide sequences of SEQ ID NO: 7(PCNA subunit) or SEQ ID NO: 9, 11, or 13 (RFC subunit).

Alternatively, the DNA encoding each of the subunits composing the clamp(PCNA) or the clamp loader (RFC) of the present invention may alsoinclude a DNA comprising a nucleotide sequence having at least 80%,preferably at least 90%, more preferably 95%, still more preferably 99%identity with the respective nucleotide sequence of SEQ ID NO: 7 (PCNA)or SEQ ID NO: 9, 11, or 13 (RFC subunit).

The present invention also relates to a recombinant vector into whichthe DNA of the present invention has been incorporated. As used herein,the “recombinant vector” refers to a vector engineered to incorporatethe DNA of the present invention into it by the recombinant DNAtechnology well known to person skilled in the art, including anexpression vector. The “expression vector” refers to a DNA constructcomprising the DNA of the present invention together with a nucleotidesequence(s) that can regulate the expression of the DNA of the presentinvention. “Regulatory sequences” are nucleotide sequences capable ofregulating the expression of the DNA of the present invention,including, for example, a promoter, an enhancer, a polyadenylationsignal, a replication initiation site, a ribosome-binding site or aShine-Dalgarno sequence, a terminator, and the like. Preferredregulatory sequences are a sequence containing a promoter and a sequencecontaining a terminator. Preferably, such regulatory sequences are fromthe host organism.

Examples of vectors include a plasmid, a cosmid, a phage, a phagemid,BAC, YAC, a virus, and the like. The preferred vector is a plasmid.

The present invention further relates to a host cell comprising theabove recombinant vector. Such a host cell is a transformed cell or atransformant, namely a host cell into which the above recombinant vectoris introduced. Examples of host cells include cells derived frommicroorganisms such as bacteria and fungi, insect cells, and mammaliancells. The preferred host is a microorganism. Preferred microorganismsinclude E. coli and Saccharomyces.

The recombinant DNA technology employed in the present inventionincludes, for example, techniques as described in Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989; Ausubel, et al., Current Protocols in Molecular Biology,John Wiley & Sons, 1998, etc.

In the presence of PCNA and RFC, the primer extension activity of theheterodimer DNA polymerase is enhanced compared to that in the absencethereof. In particular, this effect is significant at a high saltconcentration such as 200 mM NaCl. DNA, thereby, can be amplifiedwithout desalting directly from blood, body fluid, etc. Of course, DNAcan also be synthesized from a sample containing no salt. Therefore,when the present invention is used in a practical application, noconsideration is required for a salt concentration in a sample.

Further, when the RFC (the clamp loader) of the present invention loadsthe clamp on the DNA, adenosine triphosphate (ATP) is not required as anenergy source. The enzyme reaction system of the present invention, inwhich the above heterodimer DNA polymerase coexists with PCNA and RFC,does not require the addition of adenosine triphosphate (ATP) as anenergy source for loading the clamp on DNA in DNA synthesis. This is afurther characteristic, because ATP inhibits the action of these DNAreplication factors that enhance the DNA polymerase activity at a highsalt concentration.

Any of the heterodimer DNA polymerase, PCNA and RFC of the presentinvention is thermostable, and the reagent kit for DNA synthesiscomprising them in combination is suitable as a PCR reagent kit.Further, in the reagent kit, dATP, dTTP, dGTP and dCTP may beincorporated as substrates, and Family B DNA polymerase (Pol B) and thelike may also be incorporated.

The reagent kit for synthesizing a DNA of the present inventioncomprises: a DNA polymerase from Pyrococcus horikoshii as well as (a) aprotein complex composed of three molecules of a subunit and having aclamp function, wherein the subunit is a protein comprising the aminoacid sequence of SEQ ID NO: 8 or an amino acid sequence having at least90% identity with the amino acid sequence of SEQ ID NO: 8; and (b) aprotein complex composed of one molecule of a large subunit and fourmolecules of a small subunit and having a clamp loader function, whereinthe large subunit is a protein comprising the amino acid sequence of SEQID NO: 10 or an amino acid sequence having at least 90% identity withthe amino acid sequence of SEQ ID NO: 10, and the small subunit being aprotein comprising the amino acid sequence of SEQ ID NO: 14 or an aminoacid sequence having at least 90% identity with the amino acid sequenceof SEQ ID NO: 14. In the reagent kit of the present invention, each ofthe DNA polymerase, the protein complex (a), and the protein complex (b)may be contained in separate containers. In addition, in the reagent kitof the present invention, all of the DNA polymerase, the protein complex(a), and the protein complex (b) may be contained in the same container,or may be contained in any combination thereof.

Example 1 (1) The Culture of Archaeon

The archaeon JCM 9974 was cultured by the following procedure.

NaCl (13.5 g), Na₂SO₄ (4 g), KCl (0.7 g), NaHCO₃ (0.2 g), KBr (0.1 g),H₃BO₃ (30 mg), MgCl₂.6H₂O (10 g), CaCl₂ (1.5 g), SrCl₂ (25 mg), 1.0 mlof a resazurin solution (0.2 g/L), yeast extract (1.0 g), and bactopeptone (5 g) were dissolved in water to give 1 L of a solution, pH wasthen adjusted to 6.8 followed by high-pressure sterilization. To thesterilized solution, then, the element sulfur sterilized by dry heat wasadded at the final concentration of 0.2%. The resulting medium wassaturated with argon to render the medium anaerobic, into which JCM 9974was then inoculated. When a solution of Na₂S was added to the medium,the color of resazurin did not change into pink by Na₂S in the medium,and thereby the anaerobic condition of the medium was confirmed. Thismedium was incubated at 95° C. for 2 to 4 days followed bycentrifugation to collect the microorganisms.

(2) The Preparation of Chromosomal DNA

The chromosomal DNA of JCM 9974 was prepared by the following procedure.After the incubation was completed, the microorganism was collected bycentrifugation at 5,000 rpm for 10 min. The collected microorganism waswashed with a 10 mM Tris solution (pH 7.5) containing 1 mM EDTA twice,and then entrapped into a block of InCert Agarose (from FMC). This blockwas treated in a 1% N-lauroylsarcosine solution containing 1 mg/mlProtease K to separate and prepare the chromosomal DNA in the Agaroseblock.

(3) The Preparation of Clone Libraries Carrying the Chromosomal DNA

The chromosomal DNA obtained in the above (2) was subjected to partialdigestion using the restriction enzyme HindIII followed by agarose gelelectrophoresis to give a DNA fragment of about 40 kb in length. ThisDNA fragment was ligated, by using T4 ligase, with the Bac vectorpBAC108L or the fosmid vector pFOS1 completely digested using therestriction enzyme HindIII. When the former vector was used, the DNA wasintroduced into E. coli immediately after the ligation byelectroporation. When the latter vector pFOS1 was used, the DNA afterthe ligation was packaged into lambda phage particles in vitro usingGIGA Pack Gold (from Stratagene), and E. coli was then infected withthis packaged particles in order to introduce the DNA into the E. coli.The resulting E. coli population resistant to the antibioticchloramphenicol was used as the BAC library or fosmid library. Fromthese libraries, clones suitable for covering the chromosome of JCM 9974were selected for clone alignment.

(4) The Nucleotide Sequencing for the BAC or Fosmid Clone

The nucleotide sequences were determined for the aligned BAC or fosmidclone by the following steps. The DNA of the BAC or fosmid clonecollected from E. coli was sonicated to obtain DNA fragments, which werethen subjected to agarose gel electrophoresis to give 1 kb or 2 kb DNAfragments. These DNA fragments were inserted into plasmid vector pUC118at the HincII restriction enzyme site to prepare 500 shotgun clones perBAC or fosmid clone. The nucleotide sequence of each shotgun clone wasdetermined by using an automatic DNA sequencer, Type 373 or Type 377(PerkinElmer ABI). The nucleotide sequences obtained from the respectiveshotgun clones were assembled and edited by using an automatic DNAsequence assembly and analysis software, Sequencher, to determine thefull-length nucleotide sequence for the BAC clone or the fosmid clone.

(5) The Identification of Genes Encoding DNA Polymerase, Clamp and ClampLoader

The nucleotide sequences of the BAC clone and the fosmid clone asdetermined above were analyzed by a large-scale computer, and thefollowing genes were identified: the genes encoding the large subunit(underlined is an intein sequence in FIG. 1; SEQ ID NO: 1) and the smallsubunit (SEQ ID NO: 5) of the DNA polymerase; the gene encoding theclamp (PCNA) subunit (SEQ ID NO: 7); and the genes encoding the largesubunit (SEQ ID NO: 9) and the small subunit (underlined is an inteinsequence in FIG. 3; SEQ ID NO: 11) of the clamp loader (RFC).

Example 2 (1) The Construction of an Expression Plasmid for the SmallSubunit of the DNA Polymerase D

In order to form restriction enzyme (NdeI and BamHI) sites upstream anddownstream of the region of the structural gene coding for the smallsubunit (SEQ ID NO: 5), DNA primers were synthesized. Using them, therestriction enzyme sites were inserted upstream and downstream of thegene by PCR.

Upper primer: PolS1; (SEQ ID NO: 15)5′-TTTTGTCGACGTACATATGGATGAATTCGTAAAG-3′ (NdeI site is underlined)Lower primer: PolS2; (SEQ ID NO: 16)5′-TTTTGAGCTCTTTGGATCCTTAGAAGCTCCATCAGCACCACCT-3′(BamHI site is underlined)

After PCR, the extended strands were completely digested (at 37° C. for2 hr) using the restriction enzymes (NdeI and BamHI), followed bypurification of the structural gene. Further, pET11a or pET15b (Novagen)was cleaved using the restriction enzymes NdeI and BamHI and purified,which was then ligated with the above structural gene using T4 ligase at16° C. for 2 hr. A part of the ligated DNA was introduced into competentE. coli XL1-BlueMRF′ cells to obtain colonies of transformants. From theresulting colonies, expression plasmids were purified by alkaline lysis.The resulting expression plasmid was abbreviated as pET11a/PolS orpET15b/PolS, respectively. Absence of a random mutation on the structuregene was confirmed by DNA sequencing.

(2) The Construction of an Expression Plasmid for the Large Subunit ofthe DNA Polymerase D

The gene coding for the large subunit was cloned into a pGEMEX-1 vector(from Promega) by a two-step procedure. The DNA fragment of upper partof said gene was obtained by PCR using the following two primers.

Upper primer: PolL1; (SEQ ID NO: 17)5′-CTCGACTTTAGCATATGGCTCTGATGGAGC-3′ (NdeI site is underlined)Lower primer: PolL2; (SEQ ID NO: 18)5′-GCTTGTCGACGCCATAAACTTTGACATTATCCATTGCGCGCTTAAG CAAC-3′(SalI site is underlined)

The PCR products were completely digested using NdeI and SalI, and thencloned into a pGEMEX-1 vector, abbreviated as pGEM/PolL1-2.

The DNA fragment of lower part of said gene was obtained by PCR usingthe following two primers.

Upper primer: PolL3; (SEQ ID NO: 19) 5′-TTTATGGCGTCGACAAGCTGAAGG-3′(SalI site is underlined) Lower primer: PolL4; (SEQ ID NO: 20)5′-TATAACTTATGCATTGTGGTTATTTCGCTGAGAAG-3′ (Nsil site is underlined)

The PCR products were completely digested using SalI and NsiI and thencloned into the previously prepared pGEM/PolL1-2 to obtain pGEM/PolLcarrying the full-length gene coding for the large subunit.

(3) The Construction of an Expression Plasmid for the Large Subunitwithout Intein

As shown in FIG. 1, the gene coding for the large subunit of the DNApolymerase D from P. horikoshii contains one intein (coding for aproteinous intron). Therefore, the DNA fragment upstream of the inteinwas amplified by PCR using the primers PolL3 and PolL6, and the DNAfragment downstream of the intein was amplified by PCR using the primersPolL5 and PolL4. The DNA fragment without the intein was amplified byOverlap PCR using these two fragments and the primers PolL3 and PolL4.The products were then completely digested using the restriction enzymesSalI and NsiI, which were then cloned into the previously preparedpGEM/PolL1-2 to obtain pGEM/PolL(-Intein) carrying the gene coding forthe large subunit without intein (SEQ ID NO: 3).

PolL5: (SEQ ID NO: 21) 5′-CACGCTGCAAAGAGGAGAAATTGCGATGGTGATGAAGATGCT-3′PolL6: (SEQ ID NO: 22) 5′-AGCATCTTCATCACCATCGCAATTTCTCCTCTTTGCAGCGTG-3′(4) The Construction of a Plasmid Coexpressing the Small Subunit and theLarge Subunit without Intein

In order to produce the heterodimer DNA polymerase D of the presentinvention in a stable manner, a plasmid coexpressing the both subunitswas constructed. First, in order to introduce a new multi-cloning siteat the immediate upstream of the BamHI site of pET15b/PolS, PCR wasperformed using the primers PolS1 and PolS3. Herein, as described below,the BamHI, NsiI, SalI, and SacII sites were coded in PolS3 from the5′-terminal in this order. The resulting PCR products were treated withNdeI and BamHI, and then inserted into pET15b to constructpET15b/PolS(M) containing the multi-cloning site between the stop codonof the small subunit and the BamHI site.

Next, PCR was performed using the primers PolL7 and PolL2 withpGEM/PolL(-Intein) as a template. The resulting products contained a newSacII site at the 5′-terminal, and contained the portion from theribosome-binding site to the SalI site in the coding region of theprotein expression unit of pGEM/PolL(-Intein). This products weretreated with SacII and SalI, and then inserted into the multi-cloningsite generated in pET15b/PolS(M). This plasmid was abbreviated aspET15b/PolSL1-2. On the other hand, pGEM/PolL(-Intein) was treated withSalI and NsiI to isolate the lower part of the gene of the large subunit(without the intein), which then was inserted into the proteinexpression unit of the pET15b/PolSL1-2 at the remaining SalI and NsiIsites thereof. The resulting plasmid was abbreviated aspET15b/PolSL(-Intein).

This expression plasmid, pET15b/PolSL(-Intein) enables the coexpressionof the small subunit with a histidine tag attached at the N-terminalthereof and the large subunit without intein.

PolS3: (SEQ ID NO: 23) 5′-CG GGATCC ATGCAT G GTCGAC A CCGCGG TCAGCACCACCTACTAAAGTCGAG-3′ (BamHI, NsiI, SalI, and SacII sites from 5′-terminal in this order are underlined) PolL7: (SEQ ID NO: 24)5′-GGTGTCCGCGGCTCACTATAGGGAGACCAC-3′ (SaclI site is underlined) 

Example 3

The Construction of an Expression Plasmid for the Clamp (PCNA)

In order to introduce restriction enzyme (NdeI and XhoI) sites upstreamand downstream of the region of the structural gene coding for PCNA (SEQID NO: 7), DNA primers were synthesized. Using them, the restrictionenzyme sites were introduced upstream and downstream of the gene by PCR.

Upper primer: PCNA1; (SEQ ID NO: 25)5′-GGGGGCATATGCCATTCGAAATAGTCTTTGAGGG-3′ (NdeI site is underlined)Lower primer: PCNA2; (SEQ ID NO: 26) 5′-GGGGGCTCGAGTCACTCCTCAACCCTTGG-3′(XhoI site is underlined.) 

After PCR, the extended strands were completely digested (at 37° C. for2 hr) using the restriction enzymes (NdeI and XhoI), followed bypurification of the fragment of the structural gene. Further, pET11a′,in which the XhoI site was added to the multi-cloning site of pET11a,was newly constructed, which was then cleaved using the restrictionenzymes NdeI and XhoI and purified. The purified fragments were thenligated with the above structural gene using T4 ligase at 16° C. for 2hr. A part of the ligated DNA was introduced into competent E. coliXL1-BlueMRF′ cells to obtain colonies of transformants. From theresulting colonies, expression plasmids were purified by alkaline lysis.The resulting expression plasmid was abbreviated as pET11a′/PCNA.Absence of random mutation in the structure gene was confirmed by DNAsequencing.

Example 4 (1) The Construction of an Expression Plasmid for the SmallSubunit of the Clamp Loader (RFC)

As shown in FIG. 3, the gene (RFCS) coding for one of the subunitscomposing the clamp loader derived from P. horikoshii contains oneintein (coding for a proteinous intron; the underlined part in FIG. 3).Therefore, the DNA fragment upstream of the intein was amplified by PCRusing the primers RFCS1 and RFCS3, and the DNA fragment downstream ofthe intein was amplified by PCR using the primers RFCS4 and RFCS2. TheDNA fragment without the intein was amplified by Overlap PCR using thesetwo fragments and the primers RFCS1 and RFCS2. Then, the products werecompletely digested using the restriction enzymes NdeI and BamHIfollowed by the purification of the structural gene (SEQ ID NO: 13).Further, pET11a (from Novagen) was cleaved using the restriction enzymesNdeI and BamHI followed by purification. The purified fragments werethen ligated with the above fragment of the structural gene using T4ligase at 16° C. for 2 hrs. Competent cells of E. coli XL1-BlueMRF′ wasintroduced with an aliquot of the ligated DNA to obtain colonies oftransformants. From the resulting colonies, expression plasmids werepurified by alkaline lysis. The resulting expression plasmid wasabbreviated as pET11a/RFCS.

RFCS1: (SEQ ID NO: 27) 5′-GGGGGGCATATGCATAATATGGAAGAGGTTCGCGAGG-3′(NdeI site is underlined) RFCS2: (SEQ ID NO: 28)5′-GGGGGATCCTCACTTCTTCTTTCCAACTAAGGTAAA-3′ (BamHI site is underlined) RFCS3: (SEQ ID NO: 29) 5′-GCAGGTCCTCCTGGTGTTGGAAAGACTACAGCAGCTTTAGCCCTCTCA-3′ RFCS4: (SEQ ID NO: 30)5′-TGAGAGGGCTAAAGCTGCTGTAGTCTTTCCAACACCAGGAGGAC CTGC-3′

(2) The Construction of an Expression Plasmid for the Large Subunit ofthe Clamp Loader (RFC)

In order to introduce restriction enzyme (NdeI and BamHI) sites upstreamand downstream of the region of the structural gene coding for the largesubunit (SEQ ID NO: 9), DNA primers were synthesized. Therewith, therestriction enzyme sites were introduced upstream and downstream of thegene using PCR.

Upper primer: RFCL1; (SEQ ID NO: 31)5′-GGGGGGCATATGCCGGATGTTCCATGGATTGAG-3′ (NdeI site is underlined)Lower primer: RFCL2; (SEQ ID NO: 32)5′-GGGGGATCCGGGGATGCATGGGGGTCGACCTAATTCTTCTTAAT AAAGTCAAAGAGTGTG-3′(BamHI site is underlined) 

After PCR, the extended strands were completely digested (at 37° C. for2 hr) using the restriction enzymes (NdeI and BamHI) followed by thepurification of the fragment of the structural gene. Further, pET15b(from Novagen) was cleaved using the restriction enzymes NdeI and BamHIand purified. The purified fragments were then ligated with the abovefragment of the structural gene using T4 ligase at 16° C. for 2 hr. Apart of the ligated DNA was introduced into competent E. coliXL1-B1ueMRF′ cells to obtain colonies of transformants. From theresulting colonies, expression plasmids were purified by alkaline lysis.The resulting expression plasmid was abbreviated as pET15b/RFCL.

(3) The Construction of a System for Coexpressing the Large and Smallsubunits of RFC

In order to construct a system for coexpressing the large and smallsubunits of RFC by employing co-transformation, the gene coding for thelarge subunit (RFCL) was inserted between the NcoI site and the BamHIsite of the multi-cloning site 2 of pACYCDuet-1 (Novagen) that iscompatible (having an Ori sequence from a different origin) with a pETvector. The detailed procedure is explained below.

First, one NcoI recognition sequence in the RFCL gene was subjected tosilent mutation using Overlap PCR. More specifically, using pET15b/RFCLas a template, the DNA fragment upstream of the NcoI recognitionsequence was amplified by PCR using the primers RFCL3 and RFCL5, and theDNA fragment downstream of the NcoI recognition sequence was amplifiedby using the primers RFCL6 and RFCL4. The DNA fragment without the NcoIrecognition sequence was amplified by Overlap PCR using these twofragments and the primers RFCL3 and RFCL4. The primers RFCL3 and RFCL4have sequences that can anneal with the upstream and the downstream ofthe multi-cloning site of the pET15b vector, respectively, and hence theNcoI site and the BamHI site derived from the pET15b were contained inthe Overlap PCR products. These products were completely digested usingthe restriction enzymes NcoI and BamHI followed by the purification ofthe fragment of the structural gene. On the other hand, pACYCDuet-1 wascleaved using the restriction enzymes NcoI and BamHI, followed bypurification. The purified products were then ligated with the abovefragment of the structural gene using T4 ligase at 16° C. for 2 hr.Competent cells of E. coli XL2-Blue were introduced with a part of theligated DNA to obtain colonies of transformants, which were resistant tochloramphenicol at the final concentration of 500 μg/ml.

From the resulting colonies, expression plasmids were purified byalkaline lysis. The resulting expression plasmid was abbreviated aspACYC/RFCL.

(SEQ ID NO: 33) RFCL3: 5′-GCAAGGAATGGTGCATGCAAGGAGATGGCG-3′(SEQ ID NO: 34) RFCL4: 5′-AGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTT-3′(SEQ ID NO: 35) RFCL5: 5′-CCTGTACTTCTCAATCCAGGGAACATCGGGCAT-3′(SEQ ID NO: 36) RFCL6: 5′-ATGCCCGATGTTCCCTGGATTGAGAAGTACAGG-3′

Example 5 Expression of the Recombinant Genes (1) The DNA Polymerase D

Competent E. coli cells (BL21-CodonPlus (DE-3)-RIL; from Stratagene)were thawed, 0.1 ml of which was transferred into a centrifuge tube. Tothe thawed cells, 0.005 ml of a solution of the expression plasmid wasadded and kept on ice for 30 min. Then, the cells were subjected to heatshock at 42° C. for 30 sec, to which 0.9 ml of SOC medium was then addedfollowed by shaking at 37° C. for 1 hr. The cultured cells in anappropriate amount were plated onto a 2YT agar plate containingampicillin, which was incubated at 37° C. overnight to obtaintransformants.

The transformants were cultured in 2YT medium (2 L) containingampicillin until the absorbance at 600 nm reached to 1, and then IPTG(isopropyl-β-D-thiogalactopyranoside) was added thereto and furthercultured at 30° C. for 8 hr, followed by centrifugation (6,000 rpm for20 min.) to collect the microorganisms.

(2) The Clamp (PCNA)

Competent E. coli cells (BL21-CodonPlus (DE-3)-RIL; from Stratagene)were thawed, 0.1 ml of which was transferred into a centrifuge tube. Tothe thawed cells, 0.005 ml of a solution of the expression plasmid wasadded and kept on ice for 30 min. Then, the cells were subjected to heatshock at 42° C. for 30 sec., to which then 0.9 ml of SOC medium wasadded followed by shaking at 37° C. for 1 hr. Subsequently, the culturedcells in an appropriate amount were plated onto a 2YT agar platecontaining ampicillin, which was incubated at 37° C. overnight to obtaintransformants. The transformant was named E. coli BL21(DE3) CodonPlusRIL/pET11a′/PCNA and deposited under the terms of the Budapest treatywith the International Patent Organism Depositary National Institute ofAdvanced Industrial Science and Technology (AIST Tsukuba Central 6,1-1-1 Higashi, Tsukuba, Ibaraki, Japan) on Mar. 9, 2007, the assignedAccession Number being FERM BP-10796, which deposit was transferred fromthe national deposition under Accession Number FERM P-20911 (depositedon May 12, 2006).

The transformants were cultured in 2YT medium (2 L) containingampicillin until the absorbance at 600 nm reached to 0.5, and then IPTG(at the final concentration of 0.5 mM) was added thereto and furthercultured at 37° C. for 4 hr, followed by centrifugation (6,000 rpm for20 min.) to collect the microorganisms.

(3) The Clamp Loader (RFC)

The large and small subunits of RFC were co-expressed byco-transformation using the two expression vectors. First, competent E.coli cells (BL21 (DE-3); from Stratagene) were thawed, 0.1 ml of whichwas transferred into a centrifuge tube. To the thawed cells, 0.005 ml ofa solution of the expression plasmid pET11a/RFCS was added and kept onice for 30 min. Then, the cells were subjected to heat shock at 42° C.for 30 sec, to which then 0.9 ml of SOC medium was added followed byshaking at 37° C. for 1 hr. Subsequently, the cultured cells in anappropriate amount were plated onto a 2YT agar plate containingampicillin, which was incubated at 37° C. overnight to obtaintransformants, E. coli BL21 (DE3)/pET11a/RFCS. The transformants werefurther treated with CaCl₂ to prepare competent cells. To the resultingcompetent cells, 0.005 ml of a solution of the expression plasmidpACYC/RFCL was added and kept on ice for 30 min. Then, the cells weresubjected to heat shock at 42° C. for 30 sec., to which then 0.9 ml ofSOC medium was added followed by shaking at 37° C. for 1 hr.Subsequently, the cultured cells in an appropriate amount were platedonto a 2YT agar plate containing two antibiotics, ampicillin andchloramphenicol (at the final concentrations of 100 μg/ml and 50 μg/ml,respectively), which was incubated at 37° C. overnight to obtain thetransformant E. coli BL21(DE3)/pET11a/RFCS/pACYC/RFCL. This transformantwas deposited under the terms of the Budapest treaty with theInternational Patent Organism Depositary National Institute of AdvancedIndustrial Science and Technology (AIST Tsukuba Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan) on March 9, 2007, the assignedAccession Number being FERM BP-10797, which deposit was transferred fromthe national deposition under Accession Number FERM P-20912 (depositedon May 12, 2006).

The transformants were cultured in 2YT medium (200 ml) containingampicillin and chloramphenicol until the absorbance at 600 nm reached to0.6, and then IPTG (at the final concentration of 0.3 mM) was addedthereto and further cultured at 25° C. for 20 hrs. followed bycentrifugation (6,000 rpm for 20 min.) to collect the microorganisms.

Example 6 The Purification of Recombinant Proteins (1) The ThermostableDNA Polymerase D

The collected microorganisms were frozen at −20° C. and thawed, to whichthen twice the volume of 10 mM Tris HCl buffer (pH 8.0) and 1 mg ofDNase to prepare suspension. The resulting suspension was incubated at37° C. for 30 min., followed by sonication for 10 min. The suspensionwas further heated at 85° C. for 30 min. followed by centrifugation(11,000 rpm for 20 min.) to obtain the supernatant. This was used as asolution of the crude enzyme. Next, this solution of the crude enzymewas added to a Ni-column (from Novagen; a His-Bind metal chelation resin& His-Bind buffer kit were used) to carry out affinity chromatography.The 60 mM imidazole eluted fraction obtained thereby was transferredinto 100 mM phosphate buffer (pH 6.0) using a Centriprep 30 (fromAmicon). Further, this solution was adsorbed in a HiTrap SP column (fromPharmacia) and was then subjected to NaCl gradient elution. Next,SDS-PAGE was done for each fraction to determine a molecular weight of aprotein contained therein. The subunits of the DNA polymerase of thepresent invention were expected to have a molecular weight of 144,000 Daand 70,000 Da, respectively. Hence, fractions having these molecularweights were collected, which were transferred into 50 mM Tris HClbuffer (pH 7.0) using a Centriprep 30 followed by further affinitychromatography using a HiTrap Heparin column (from Pharmacia) and NaClgradient elution to obtain a purified enzyme.

(2) The Thermostable Clamp (PCNA)

The collected microorganisms were frozen at −20° C. and thawed, to whichthen twice the volume of 50 mM Tris HCl buffer (pH 8.0), 0.1 M NaCl, 2mM 2-mercaptoethanol, 0.1 mM EDTA, and 10% glycerol to preparesuspension. The resulting suspension was subjected to sonication for 10min. followed by centrifugation (30,000 g for 20 min.) to obtain thesupernatant. This was further heated at 75° C. for 15 min. followed bycentrifugation (30,000 g for 20 min.) to obtain the supernatant. Thiswas still further heated at 80° C. for 10 min. followed bycentrifugation to obtain the supernatant. Next, to the resultingsupernatant, poly(ethyleneimine) (Sigma) and NaCl were added at thefinal concentrations of 0.15% and 0.58 M, respectively, which was thenstirred at 4° C. for 30 min followed by centrifugation (30,000 g for 20min.). To the supernatant, (NH₄)₂SO₄ was added until 80% saturation,which was then stirred on ice for 2 hrs. followed by centrifugation(30,000 g for 20 min) to recover the precipitate. This precipitate wasdissolved in 50 mM Tris HCl buffer (pH 8.0), 0.1 M NaCl, 2 mM2-mercaptoethanol, 0.1 mM EDTA, and 10% glycerol. This solution wasdialyzed with the same buffer and added to an anion exchange column,HiTrap Q (from Amersham Pharmacia; 5 ml), which had been equilibratedwith 50 mM Tris HCl buffer (pH 8.0), followed by from 0 to 1 M NaClgradient elution in the same buffer. Further, the resulting solution wasadded to a Superdex 200 column (10/300; Amersham Pharmacia), which hadbeen equilibrated with 50 mM Tris HCl buffer (pH 8.0) and 0.2 M NaCl,followed by elution with the same buffer to obtain purified PCNA.

(3) The Thermostable Clamp Loader (RFC)

The collected microorganisms were frozen at −20° C. and thawed, to whichthen twice the volume of 50 mM Tris HCl buffer (pH 8.0) containing aprotease inhibitor (Complete, EDTA-free; from Roche) was added toprepare a suspension. The resulting suspension was homogenized using aFrench press, and then heated at 75° C. for 15 min. followed bycentrifugation (30,000 g for 20 min) to obtain the supernatant. Thissupernatant was added to a HiTrap heparin column (from AmershamPharmacia; 5 ml), which had been equilibrated with 50 mM Tris HCl buffer(pH 8.0), followed by from 0 to 1 M NaCl gradient elution in the samebuffer. The eluted fractions were analyzed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) to identify the elution peak of the RFCcomplex composed of the large and small subunits. Further, the fractionshaving these elution peaks were collected and added to a Ni-column (fromNovagen; a His-Bind metal chelation resin & His-Bind buffer kit wereused) to carry out affinity chromatography. The 200 mM imidazole elutedfraction obtained thereby was concentrated using a Centriprep 30(Amicon). This concentrated product was added to a Superdex 200 column(10/300; Amersham Pharmacia), which had been equilibrated with 50 mMTris HCl buffer (pH 8.0) and 0.2 M NaCl, followed by elution with thesame buffer to obtain the purified RFC complex.

Example 7 The Evaluations of the Reaction System of the DNA Polymerase(1) The Test Conditions (a) PCR

In order to detect the activity of the DNA polymerase D of the presentinvention, PCR was performed using the aforementioned two DNA oligomers(Upper primer and Lower primer), and the expression vector pET15b/PolSencoding the small subunit of the DNA polymerase of the presentinvention as a template DNA. One cycle was composed of three steps (94°C. for 1 min.; 61° C. for 2 min.; 70° C. for 3 min.), and 35 cycles wererepeated. The reaction solution (100 μl) contained 20 mM Tris HCl buffer(pH 8.8), 10 mM KCl, 4 mM MgSO₄, 0.1% Triton X-100, 0.375 mM dNTP mix,100 pmol Upper primer, 100 pmol Lower primer, and 0.1 μg of the DNApolymerase.

(b) DNA Synthesis

DNA synthesis was based on the method by Kornberg, et al. (J. Biol.Chem., 237, 519-525; J. Biol. Chem., 236, 1487-1493). The reactionsolution (200 μl) contained 20 mM Tris HCl buffer (pH 8.8), 10 mM KCl,10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1% Triton X-100, 0.25 mM dNTP mix, 0.37Mbq (α-³²P) dATP, 20 μg of salmon testicular DNA treated by heating andchilling, and 0.1 μg of the DNA polymerase. The reaction was performedat 75° C. for 30 min. After the reaction was completed, to the reactionsolution, 0.5 mg of the salmon testicular DNA chilled on ice was added,and thereto 500 μl of 1 N perchloric acid and 500 μl of water, bothchilled on ice, were added followed by centrifugation (9,000×g for 5min.) to obtain an acid-insoluble fraction. This precipitate wasdissolved in 300 μl of 0.2 N NaOH, and further thereto 300 μl of 1 Nperchloric acid and 300 μl of water, both chilled on ice, were addedfollowed by centrifugation to obtain an acid-insoluble fraction. Thisprecipitate was washed with 1 ml of 1 N acetic acid followed bycentrifugation. The resulting precipitate was dissolved in 0.4 ml of 2 Nammonia solution. The radioactivity was determined by the Cherenkoveffect using a liquid scintillation counter. Herein, the amount of theenzyme that can incorporate 10 nmol of dNTPs in DNA synthesis at 75° C.for 30 min. is defined as 1 Unit.

(c) The Optimum pH

The optimum pH was determined by incorporation of radioactivity into theacid-insoluble fraction, wherein the reaction temperature was held at75° C. under the above test conditions and pH was changed from 5.8 to9.5 with a phosphate buffer in the acidic range and with a Tris HClbuffer in the basic range.

(d) The Optimum Concentration of Mg²⁺

The optimum concentration of Mg²⁺ was determined by the incorporation ofradioactivity into the acid-insoluble fraction, wherein the reactiontemperature was fixed at 75° C. under the above test condition and theconcentration of MgSO₄ was changed from 0 mM to 20 mM.

(e) Thermostability

The enzyme solution (100 μl) for heating contained 20 mM Tris HCl buffer(pH 8.0 at 25° C.), 500 mM NaCl, 10 mM MgSO₄, and the enzyme of thepresent invention at a concentration of 0.1 mg/ml. This solution washeated for 1 hr. in the range of from 60° C. to 95° C. using a GeneAmpPCR System 2400 (PerkinElmer), and then, with the resulting solution,DNA synthesis was performed and the residual activity was determined bythe incorporation of radioactivity into the acid-insoluble fraction.

(f) The Primer Extension Activity

The primer extension activity was determined by the following procedure.A single-strand DNA of M13 phage (0.2 μg) and each of the primers (0.5pmol) (15 mer, 34 mer, and 50 mer), which had been 5′-labeled with ³²P,were annealed in 20 mM Tris HCl buffer (pH 8.5), 0.05 Units of the DNApolymerase was added to the DNA mixture in the presence of 10 mM MgCl₂,and then the reaction was allowed to proceed at 75° C. The reaction wasterminated after 2 min or 10 min by adding the Stop solution thereto.The reaction products were analyzed by electrophoresis with 15%polyacrylamide gel (PAGE) containing 8 M urea.

(g) The 3′-5′ Exonuclease Activity

By using the 50 mer primer 5′-labeled with ³²P (0.5 pmol), the 3′-5′exonuclease activity was determined. The reaction solution (20 μl)contained 20 mM Tris HCl buffer (pH 8.5), 12 mM MgCl₂, and 4 ng of thelabeled DNA, to which 0.05 Units of the polymerase was added, and thereaction was allowed to proceed at 75° C. The reaction was terminatedafter 30 min. by adding the Stop solution thereto. The reaction productswere analyzed by electrophoresis with 15% polyacrylamide gel (PAGE)containing 8 M urea.

(h) The Interaction between RFC Complex and PCNA

The binding property of RFC complex and PCNA was examined by thefollowing procedure. In 100 μl of the reaction solution (containing 10mM MgCl₂, 0.5 M NaCl, and ATP at a predetermined concentration), 400pmol of PCNA and 400 pmol of RFC complex were admixed and kept at roomtemperature for 20 min. Thereto, 40 μl of Ni²⁺-chilating resin was addedand stirred by shaking at room temperature for 10 min, followed bycentrifugation at 5,000 g for 30 sec to recover the resin as theprecipitate. This recovered resin was washed with 200 μl of 20 mM TrisHCl buffer (pH 8.0) containing 0.5 M NaCl, 10 mM MgCl₂, and 5 mMimidazole four times followed by elution of the target protein complexusing 40 μl of 20 mM Tris HCl buffer (pH 8.0) containing 0.5 M NaCl, 10mM MgCl₂, and 500 mM imidazole. The composition of the proteins in theeluted samples was analyzed by SDS-PAGE.

(i) The Effect of RFC Complex and PCNA on the DNA Polymerase Activity

The primed substrate, which would be a substrate of the DNA polymeraseD, was prepared by the following procedure. First, the 5′-terminal ofthe 51 mer oligonucleotide(5′-GTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAAGGACGGCCAGTGCC-3′ (SEQ ID NO:37)) was labeled with ³²P. The reaction solution (20 μl) contained 1×kinase buffer (from Toyobo), 20 pmol of the above 51 meroligonucleotide, 6 μl of γ-³²P ATP (3,000 Ci/mmol), and 2 μl of T4Kinase. This reaction solution was allowed to react at 37° C. for 3 hrs.followed by heating at 95° C. for 3 min. The oligonucleotide labeledwith ³²P was purified using QIAquick Nucleotide Removal Kit (QIAGEN).Next, in 100 μl of 1× M buffer (from Toyobo), 2 nmol of M13 ssDNA and 4pmol of the 51 mer oligonucleotide labeled with ³²P were admixed andthen boiled for 5 min. followed by allowing the solution to coolgradually for annealing.

Using the above primed M13 ssDNA as a template, DNA polymerase reactionwas carried out. To the reaction system, RFC complex and PCNA wereadded, and thereby the effect of these DNA replication factors toenhance the DNA polymerase activity was evaluated. The composition andthe final concentrations of the reaction solution (20 μl) were asfollows:

20 mM Tris HCl buffer (pH 8.8)

10 mM KCl

10 mM (NH₄)₂SO₄

0.1% Triton X-100

6 mM MgSO₄

0.25 mM dNTPs

400 ng of the DNA polymerase

2 μg of PCNA

2 μg of the RFC complex

0.04 pmol of the ³²P-labeled primed M13 ssDNA

50-200 mM NaCl

10 mM ATP

This reaction solution (20 μl) was kept at 60° C. for 5 min. After thereaction was completed, 4 μl of the reaction termination solution (0.3 MNaOH, 60 mM EDTA, 0.1% Bromophenol Blue, and 10% glycerol) was addedthereto. This solution was then boiled for 5 min. followed by analysisof molecular species in the reaction product by 1.2% alkaline agarosegel electrophoresis, with the electrode solutions of 50 mM NaOH and 1 mMEDTA.

(2) The Results of the Evaluations (a) The Properties of the DNAPolymerase D 1) The Protein Chemical Properties

The large subunit of the enzyme of the present invention was composed of1,434 amino acid residues before the removal of the intein and 1,268amino acid residues after the removal of the intein, and the respectivemolecular weights were 163,000 Da and 144,000 Da. The small subunitthereof was composed of 623 amino acid residues, the molecular weightbeing 70,000 Da.

2) The Improved Activity of the Active Enzyme by Coexpressing the SmallSubunit and the Large Subunit without the Intein

When expressed individually, each of the subunits was extremelyunstable, and hence stable expression was not possible. Therefore, thecoexpression system was constructed. Thereby, it was possible to expressthe active enzyme at a high level, and the physicochemical propertiesthereof and the mechanism of thermostability were able to be explored.

3) The Detection of DNA Synthesizing Activity and PCR

By using a solution of the crude enzyme from recombinant E. coli, theDNA synthesizing activity was examined. As shown in Table 1, no activitywas detected with each of the subunits itself. The combination of thesmall subunit and the large subunit containing intein also exhibited noactivity. From these results, it was evident that the heterodimerstructure composed of the small subunit and the large subunit withoutintein is essential to exhibiting the activity. Further, the heterodimerenzyme pET15b/PolSL(-Intein) composed of the small subunit and the largesubunit without intein was purified. This purified enzyme (0.1 μg) and10 mM MgSO₄ were used to determine the activity to synthesize DNA. Inthe system with the enzyme, an increased radioactivity, 175 times ormore, was detected when compared to that without the enzyme. Next, thepurified enzyme of the present invention was used for PCR and it wasconfirmed that the amplified product had the same length as the targetDNA fragment (1.9 kb) by agarose gel electrophoresis. On the other hand,in the system without the enzyme of the present invention, no PCRproduct was detected. From the above findings, it was further evidentthat the heterodimer DNA polymerase D of the present invention composedof the small subunit and the large subunit without intein hassufficiently activity.

TABLE 1 The detection of the DNA synthesizing activity using a solutionof the crude enzyme (2 ml) obtained from the recombinant E. coli Enzymesource Activity (CPM) Negative control (distilled water only) 2,567pET15b/PolS 1,181 pGEM/PolL 4,777 pGEM/PolL(-Intein) 3,827 pET15b/PolSL3,237 pET15b/PolSL (-Intein) 12,323 Positive control (Deep Ventpolymerase, 1 Unit) 86,189

4) Optimum pH

The optimum pH at 75° C. was 8.5 (FIG. 5).

5) Optimum Mg²⁺ Concentration

The optimum Mg²⁺ concentration of the enzyme of the present inventionwas 12 mM (FIG. 6).

6) Thermostability

As shown in FIG. 7, the enzyme of the present invention retained 50% ofthe activity after heating at 85° C. for 1 hr. Further, it retained 20%of the activity even after heating at 90° C. for 1 hr.

7) Primer Extension Activity

As shown in FIG. 8, while no primer extension activity was detected withthe 15 mer primer by the enzyme of the present invention, when theprimer was lengthened up to, for example, 34 mer or 50 mer long, theprimer extension activity became greater. Such a primer extensionactivity dependent on primer length has not been reported with any otherDNA polymerase.

8) 3′-5′ Exonuclease Activity

As shown in FIG. 9, the enzyme of the present invention exhibited great3′-5′ exonuclease activity on the 50 mer oligonucleotide. From thesefindings, it was revealed that the enzyme of the present invention is aDNA-dependent DNA polymerase; is a heterodimer protein composed of twosubunits having molecular weights of 144 kDa and 70 kDa, respectively;uses DNA as a template to synthesize a complementary strand; and has3′-5′ exonuclease activity.

(b) Properties of PCNA

PCNA was expressed in a large quantity in recombinant E. coli. Most ofthe proteins derived from the E. coli were removed by heating, thenucleic acids were removed with polyethyleneimine, and ammonium sulfateprecipitation was carried out for concentration followed by anionexchange chromatography and gel filtration column chromatography forcomplete purification. As shown in FIG. 10, the apparent molecularweight of the PCNA subunit estimated by SDS-PAGE matched with themolecular weight of 28 kDa predicted from the gene. Further, themolecular weight of the native form thereof was predicted to be 110 kDafrom the elution position in the gel filtration chromatography of thepurified sample, and hence PCNA was revealed to be a homotrimer.

(c) Properties of RFC Complex 1) SDS-PAGE for RFC Complex

RFC complex was expressed in a large quantity in recombinant E. coli.Most of the proteins derived from the E. coli were removed by heating,and then affinity chromatography using heparin, affinity chromatographyusing nickel resin for the histidine tag present at the N-terminal ofthe large subunit, and gel filtration column chromatography were carriedout for complete purification. As shown in FIG. 11, the apparentmolecular weights of the large subunit (L) and the small subunit (S) ofRFC complex estimated by SDS-PAGE were 60 kDa and 36 kDa, respectively,which approximately matched with the respective molecular weights of 54kDa and 38 kDa predicted from the gene. Further, the molar ratio of thelarge subunit and the small subunit was 1:4, which was calculated fromthe dye-coupling intensities of the bands of the large and small subunitproteins, and hence the native form thereof was revealed to be aheteropentamer.

(d) Recognition of PCNA by RFC Complex in the Absence of ATP

PCNA that bound to RFC complex was pulled down by using the histidinetag of RFC complex and nickel resin, which then was analyzed bySDS-PAGE. The results were shown in FIG. 12. From these results, it wasrevealed that both of RFC complex and PCNA were purified as activeforms, and that RFC complex and PCNA recognize each other and have anactivity to be bound together. Further, it was shown that this bindingis not dependent on ATP.

(e) Requirement of ATP for RFC Complex and PCNA to Enhance the Activityof the DNA Polymerase at a High Salt Concentration

As shown in FIG. 13, it was revealed that while the DNA polymerase D byitself was not able to exhibit DNA polymerase activity in the presenceof 200 mM NaCl, when PCNA was added thereto, the enzyme was able tosynthesize DNA up to 0.75 kb. On the other hand, it was revealed thatwhen RFC complex and PCNA were added to the DNA polymerase D in thepresence of 200 mM NaCl, the enzyme was able to synthesize thefull-length complementary strand of M13ssDNA (7.2 kb) at 60° C. for 5min. At this time, the addition of ATP was not required. On thecontrary, it was found that, when 10 mM ATP was present, the effect ofRFC complex and PCNA to enhance the DNA polymerase activity at a highsalt concentration was significantly inhibited.

From the above results, it was revealed that the primer extensionactivity of the heterodimer DNA polymerase of the present invention at ahigh salt concentration can be significantly enhanced in the presence ofPCNA and RFC, when compared to that in the absence thereof.

1-3. (canceled)
 4. A composition comprising: a DNA polymerase fromPyrococcus horikoshii and two protein complexes of the following a) andb): a) a protein complex composed of three molecules of a subunit andhaving a clamp function, the subunit being a protein comprising theamino acid sequence of SEQ ID NO: 8 or an amino acid sequence having atleast 90% identity with the amino acid sequence of SEQ ID NO: 8; b) aprotein complex composed of one molecule of a large subunit and fourmolecules of a small subunit and having a clamp loader function, whereinthe large subunit is a protein comprising the amino acid sequence of SEQID NO: 10 or an amino acid sequence having at least 90% identity withthe amino acid sequence of SEQ ID NO: 10, and wherein the small subunitis a protein comprising the amino acid sequence of SEQ ID NO: 14 or anamino acid sequence having at least 90% identity with the amino acidsequence of SEQ ID NO:
 14. 5. The composition of claim 4, formulated asa reagent kit for synthesizing DNA using PCR. 6-20. (canceled)
 21. Thecomposition of claim 4, wherein a) is a protein complex composed ofthree molecules of a subunit and having a clamp function, the subunitbeing a protein comprising the amino acid sequence of SEQ ID NO:
 8. 22.The composition of claim 4, wherein a) is a protein complex composed ofthree molecules of a subunit and having a clamp function, the subunitbeing a protein comprising an amino acid sequence having at least 95%identity with the amino acid sequence of SEQ ID NO:
 8. 23. Thecomposition of claim 4, wherein b) is a protein complex composed of onemolecule of a large subunit and four molecules of a small subunit andhaving a clamp loader function, wherein the large subunit is a proteincomprising the amino acid sequence of SEQ ID NO:
 10. 24. The compositionof claim 4, wherein b) is a protein complex composed of one molecule ofa large subunit and four molecules of a small subunit and having a clamploader function, wherein the large subunit is a protein comprising anamino acid sequence having at least 95% identity with the amino acidsequence of SEQ ID NO:
 10. 25. The composition of claim 4, wherein b) aprotein complex composed of one molecule of a large subunit and fourmolecules of a small subunit and having a clamp loader function, whereinthe small subunit is a protein comprising the amino acid sequence of SEQID NO:
 14. 26. The composition of claim 4, wherein b) a protein complexcomposed of one molecule of a large subunit and four molecules of asmall subunit and having a clamp loader function, wherein the smallsubunit is a protein comprising an amino acid sequence having at least95% identity with the amino acid sequence of SEQ ID NO:
 14. 27. Areagent kit comprising the composition of claim 4, wherein the DNApolymerase and protein complexes (a) and (b) are contained in separatecontainers.
 28. A reagent kit comprising the composition of claim 4 andat least one of a family B DNA polymerase and one or more substrateselected from the group consisting of dATP, dTTP, dCTP, and dGTP.